Fig 1.
Nascent C-circles and 5' C-overhangs are generated during telomere replication.
(A) FACS analysis of G1/S synchronized U2OS cells. Cells were synchronized by double thymidine block, then released and harvested at the indicated time. (B) C-circle assay was performed at the indicated time after release from G1/S. (C) Representative image and statistical analysis showing that RPA2 foci colocalize with telomere at each time point. Cells with more than 5 colocalized foci/cell were scored positively, >100 cells were counted per time point. Error bars represent the mean ± SEM of three independent experiments.(D) BrdU pulse-labeling strategy. U2OS cells were synchronized at G1/S, released in presence of BrdU for 12h. (E) Leading, lagging and unreplicated telomeric fractions were resolved by CsCl gradient ultracentrifugation and hybridized with telomeric probe. Non-BrdU labeled U2OS was used as a negative control (upper figure). “Area under peak” for leading, lagging and unreplicated telomeres was analyzed by Graphpad Prism and the relative amount of telomeres was indicated above individual peak. (F) Nascent C-circle is predominantly associated with lagging strand DNA synthesis. C-circle assay analysis of CsCl gradient fractions in (E). The amount of C-circle in leading, lagging and unreplicated telomeres was calculated by determining "area under peak" using Graphpad Prism. The relative amount of C-circles was indicated above individual peak. (G) Schematic of the migration of linear dsDNA, ssDNA (C-overhangs) and telomeric open circles (T-circle) during 2D agarose gel electrophoresis and hybridization to a telomere-specific G-rich probe under native or denatured condition. (H) 5' C-overhang DNA is predominantly associated with leading strand DNA synthesis. The fractions corresponding to leading, lagging or non-replication telomeres from 12h BrdU labeled sample in (E) were pooled. DNA was incubated with or without RecJf, analyzed by 2D agarose gel electrophoresis and hybridized with G-rich telomeric probe under native and denaturing conditions. C-overhangs were indicated by red arrows. C-overhangs abundance was expressed as a ratio between the native and denatured signals. Values were then normalized with leading C-overhangs to obtain relative abundance.
Fig 2.
C-circles and 5' C-overhangs are linked to DNA damage-induced replication fork collapse.
(A) Replication fork stalling induced by HU or aphidicolin decreases abundance of C-circles in U2OS cells. U2OS cells were treated with HU (hydroxyurea, 2mM) or aphidicolin (Aphi, 1μg/mL) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. *P<0.05, ***P<0.001. (B) Treatment of U2OS cells with HU or aphidicolin does not change the abundance of 5' C-overhangs. U2OS cells were treated with HU (2mM) or aphidicolin (1μg/mL) for 24h. C-overhangs abundance was expressed as a ratio between the native and denatured signals. Values were then normalized with C-overhangs in untreated cells (Ctrl) to obtain relative abundance. Experiments were duplicated and the mean of relative abundance of C-overhangs was indicated. (C) C-circles are increased in U2OS cells treated with zeocin. U2OS cells were treated with zeocin (100μg/mL) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. ***P<0.001. (D) 5' C-overhangs in U2OS cells, are increased upon zeocin treatment compared to DMSO. RecJf digestion was used as a control. U2OS cells were treated with zeocin (100μg/mL) for 24h. C-overhangs abundance was expressed as a ratio between the native and denatured signals. Values were then normalized with C-overhangs in untreated cells (Ctrl) to obtain relative abundance. Experiments were repeated three times and the mean ± SEM was indicated. (E) CPT (camptothecin) increases C-circles in U2OS cells. U2OS cells were treated with CPT (0.25μM) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. ***P<0.001. (F) CPT increases the abundance of 5' C-overhangs in U2OS cells compared to DMSO treatment. U2OS cells were treated with CPT (0.25μM) for 24h. C-overhangs abundance was expressed as a ratio between the native and denatured signals. Values were then normalized with C-overhangs in untreated cells (Ctrl) to obtain relative abundance. Experiments were repeated three times and the mean ± SEM was indicated.
Fig 3.
Endogenous ssDNA break/gap or induced ssDNA break in C-rich strand stimulates formation of C-circles and 5' C-overhangs.
(A) Experimental protocol to study strand specific (G-rich or C-rich) breaks/gaps on telomere is shown schematically. HinfI and RsaI digested genomic DNA was purified and further digested with Exo III to examine potential breaks/gaps on G-strand or C-strand of telomeres. If breaks/gaps occur on C-strand, Exo III would degrade all C-strand, leaving single-stranded G-strand that can be detected by hybridization with C-rich probe under native or denatured condition. Contrariwise, only C-strand can be detected if breaks/gaps occur on G-strand. (B) Breaks/gaps occur more frequently on C-rich strand of telomere. Exo III digestion produces single-stranded DNA that is less in molecular weight than corresponding double-stranded DNA, thereby migrating faster during electrophoresis. (C) Methyl-methane sulfonate (MMS) stimulates formation of C-circle DNA in U2OS cells. U2OS cells were treated with MMS (0.25mM) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. ***P<0.001. (D) MMS stimulates formation 5' C-overhang DNA in U2OS cells. U2OS cells were treated with MMS (0.25mM) for 24h. C-overhangs abundance was expressed as a ratio between the native and denatured signals. Values were then normalized with C-overhangs in untreated cells (Ctrl) to obtain relative abundance. Experiments were repeated three times and the mean ± SEM was indicated. (E) Experimental protocol using CRISPR-Cas9 system to introduce ssDNA breaks at telomere is shown schematically. Cells express nuclease-deficient CRISPR-Cas9 (dCas9), wild type CRISPR-Cas9 (WT) or CRISPR-Cas9 with mutation at RuvC domain (D10A). dCas9 lacks nuclease activity, wtCas9 introduces dsDNA breaks, and Cas9 D10A introduces ssDNA breaks in C-rich strand of telomere. (F) Western blot of dCas9, wtCas9and Cas9 D10A expressed in HEK 293T cells. Cells are harvested 48h after transfection. Monoclonal ANTI-FLAG M2 antibody was used to determine expression level of flag-Cas9. β-actin was used as a loading control. (G) Effect of WT and mutant Cas9 on formation of C-circles in HEK 293T cells. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. **P<0.01. (H) Effect of WT and mutant Cas9 on formation of 5' C-overhangs in HEK 293T cells.
Fig 4.
Replication fork collapse and replication fork regression at telomeres in U2OS cells.
(A) U2OS cells were cultured in presence or absence of CPT and analyzed with IF-FISH to detect PML bodies on telomeres. (B) Quantification of (A). Cells with ≥3APBs were scored. >100 cells were counted for each experiment. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. ***P<0.001. (C) U2OS cells were treated with or without CPT and analyzed by IF-FISH to detect 53BP1 foci on telomeres. (D) Quantification of (C). Cells with ≥3 co-stained foci were scored. >100 cells were counted for each experiment. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. ***P<0.001. (E)-(L) U2OS cells were treated with or without CPT and analyzed by IF-FISH using telomeric G-rich probe and antibodies to RPA2 (E), SMARCAL1 (G, SM), Rad51 (I) or SLX4 (K), respectively. Quantification of panels (E), (G), (I), and (K) are shown in (F), (H), (J), and (L), respectively. Cells with ≥3 RPA2 (F), ≥1 SMARCAL1 (H),≥2 Rad51 (J), ≥3 SLX4 (L) foci colocalized with telomeres were scored. >100 cells were counted for each experiment. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. *P<0.05, **P<0.01.
Fig 5.
Defect in replication fork regression increases abundance of C-circles and 5' C-overhangs.
(A) Western blot shows efficiency of RPA2 knockdown by siRNA. β-actin was used as a loading control. U2OS cells were collected 60h after transfection with siRNA. (B) Abundance of C-circles in RPA2-depleted cells. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. *P<0.05, **P<0.01. (C) Abundance of 5' C-overhangs in RPA2-depleted cells. C-overhangs abundance was expressed as a ratio between the native and denatured signals. Values were then normalized with C-overhangs in control cells (siNC) to obtain relative abundance. Experiments were repeated three times and the mean ± SEM was indicated. (D) Western blot shows efficiency of SMARCAL1 (SM) knockdown by siRNA. β-actin was used as a loading control. U2OS cells were collected 60h after transfection with siRNA. (E) Abundance of C-circles in SMARCAL1-depleted cells. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. **P<0.01. (F) Abundance of 5' C-overhangs in SMARCAL1-depleted cells. C-overhangs abundance was expressed as a ratio between the native and denatured signals. Values were then normalized with C-overhangs in control cells (siNC) to obtain relative abundance. Experiments were duplicated and the mean of relative abundance of C-overhangs was indicated. (G) Effects of Rad51 inhibitor B02 on abundance of C-circles. U2OS cells were treated with B02 (27.4μM) for 24h. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. ***P<0.001. (H) Effects of Rad51 inhibitor B02 on abundance of 5' C-overhangs. C-overhangs abundance was expressed as a ratio between the native and denatured signals. Values were then normalized with C-overhangs in control cells (siNC) to obtain relative abundance. Experiments were repeated four times and the mean ± SEM was indicated.
Fig 6.
Telomeric HR is associated with replication fork regression.
(A) Representative images showing C-strand (red) and G-strand (green) of telomeres on sister chromatins that are visualized by CO-FISH (chromosome orientation fluorescence in situ hybridization) assay. Yellow spot representing the occurrence of T-SCE was indicated by yellow arrows. (B) Representative images showing T-SCEs in U2OS cells depleted for SMARCAL1. (C) Quantification of (B). The number of chromosomes scored (n) in three independent experiments is indicated. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. **P<0.01. (D) Representative images showing T-SCEs in U2OS cells treated with CPT. (E) Quantification of (D). The number of chromosomes scored (n) in three independent experiments is indicated. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t-test was used to calculate P-values. **P<0.01.
Fig 7.
Proposed model summarizing effects of replication fork collapse and replication fork regression on formation of C-circles and C-overhangs in ALT cells.
Replication fork collapse induced by break or gap on the C-rich strand of telomere, if not rescued, leads to different consequences for leading and lagging replication: for leading synthesis, long single-stranded C-rich DNA (C-overhang) remains unreplicated, whereas stalled lagging replication fork is cut out and cyclized to form C-circles. Fork regression machinary including SMARCAL1, SLX4 and Rad51 may restore collapsed replication fork, and thereby suppressing C-circle and C-overhang formation. HR-mediated fork regression is based on the model proposed by Petermann and Helleday [31].